Gemcitabine (2'-2'-difluorodeoxycytidine (dFdC)) is a deoxycytidine analogue that is effective against solid tumors, including lung cancer and ovarian cancer. dFdC requires the phosphorylation by deoxycytidine kinase (dCK) as a primary step in its activation. Deficiency of dCK is associated with resistance against this compound both in vitro in cancer cell lines and in clinical practice in acute myeloid leukemia and solid tumors. The human ovarian cancer cell line AG6000 is 100,000-fold resistant against dFdC compared to its parent cell line A2780. This cell line proved to be dCK deficient in enzyme activity assays and by Western blot analysis, but by RT-PCR, a normal and a truncated dCK mRNA was found. Sequencing revealed that exon 3 was deleted from the dCK cDNA, resulting in a 74-aa-long open-reading frame due to the generation of a premature stop codon. No gross genomic alteration was observed at the dCK locus, suggesting the involvement of post-transcription mechanisms. Transient transfection experiments indicated that the truncated dCK transcripts are not translated to protein. To study the functional role of the truncated dCK transcripts, both A2780 cells and AG6000 cells were stably transfected with human and rat dCK. The results indicated that over-expression of full-length dCK genes in AG6000 failed to completely reverse the sensitivity to dFdC or other drugs.